A huge numbers of organs and tissues exist in a living body of vertebrates including a human. The organs and tissues are originally formed from a single fertilized egg by undergoing cell division (cleavage) and cell differentiation, and finally constitute an individual body with a well-balanced system. Processes for the formation of organs and tissues are highly complicated, through which important intercellular interactions called induction phenomena are considered to be involved in multiple steps.
Attempts have been made for in vitro reproduction of the organ formation processes occurring in vivo to form a desired organ from undifferentiated cells (see, for example, “Organ formation from undifferentiated cells”, Inflammation/Regeneration, Vol. 22, 21, 2002 as well as Japanese Patent Unexamined Publication (Kokai) Nos. 2001-299335 and 2001-333770 for organ formation of pancreas). For example, when animal cap cells (pluripotent cell aggregates) of Triturus pyrrhogaster at the blastula stage as undifferentiated cells are treated with activin at a high concentration, a rhythmically beating heart can be formed in a formation rate of 60%. The resulting heart can maintain a normal number of beats even for one month or longer, and gene expression specific to cardiomyocytes, existence of an intercalated disc specific to cardiac muscles and the like can also be observed. Therefore, the heart is considered to a substantially complete heart from viewpoints of function and structure.
Retinoic acid (vitamin A acid) is an active metabolite of vitamin A and has extremely important physiological actions such as an action of differentiating immature cells under development to mature cells having a peculiar function, cell growth promoting action and a life-supporting action. Various vitamin A derivatives having been synthesized so far, e.g., benzoic acid derivatives described in Japanese Patent Unexamined Publication (Kokai) Nos. 61-22047 and 61-76440, compounds described in Journal of Medicinal Chemistry, 1988, Vol. 31, No. 11, p. 2182 and the like, have also been elucidated to have similar physiological actions. Retinoic acid and the aforementioned compounds having retinoic acid-like biological activities are generically referred to as “retinoids.”
It is known that retinoic acid is a regulatory factor for the embryonic patterning along the anteroposterior axis (Nature, 340, 140-144, 1989; Development, 112, 945-958, 1991; Dev. Biol., 192, 1-16, 1997; Zool. Sci., 15, 879-886, 1998), and that the retinoic acid transforms the anterior neural tissue of Xenopus embryo to a posterior one and is effective on mesodermal development (Genes Dev., 5, 175-187, 1991; Develop. Growth. Differ., 35, 123-128, 1993). It has also been reported that treatment with activin can induce most of mesodermal tissues such as notochord, muscle, mesenchyme and coelomic epithelium dose-dependently in Xenopus animal cap cells (Roux's Arch. Dev. Biol., 198, 330-335, 1990; Nature, 347, 391-394, 1990; Roux's Arch. Dev. Biol., 200, 230-233, 1991), and changing the dosage of retinoic acid that is used for co-treatment with activin enables the mesodermal tissues such as notochord, muscle and pronephros differentiated from animal cap cells to be lateralized and posteriorized (Develop. Growth. Differ., 35, 123-128, 1993).
As for the action of retinoic acid on the endodermal organ, it has been reported by Dixon et al. that when Xenopus embryos at developmental stages 22 to 32 are treated with retinoic acid, the morphology of digestive organs such as the intestines, liver and stomach becomes abnormal. However, it has also been reported that the pancreas of Xenopus embryos at developmental stages 22 to 32 treated with retinoic acid is formed normally, and no influence is found in the expression of XlHbox8, an endoderm-specific marker (Dev. Genes Evol., 208, 318-326, 1998).
It has also been revealed that all-trans retinoic acid binds to a retinoic acid receptor (RAR) belonging to the intranuclear receptor superfamily (Evans, R. M., Science, 240, p. 889, 1988), which exists in the cell nucleus, as a ligand to regulate growth/differentiation or death of animal cells (Petkovich, M., et al., Nature, 330, pp. 444-450, 1987). It is known that pancreas can be formed in vitro by using the all-trans retinoic acid or using all-trans retinoic acid and activin in combination (Japanese Patent Unexamined Publication (Kokai) Nos. 2001-299335 and 2001-333770).
As for the expression of the physiological activities of retinoic acid, existence of retinoid X receptors (RXRs, binding to 9-cisretinoic acid as a natural ligand (this compound is also serve as a ligand of RARs)) has been verified. It has been elucidated that RXR forms a dimer with RAR to induce or suppress gene transcription and thereby regulate the expression of the physiological activities of retinoic acid (Mangelsdorf, D. J. et al., Nature, 345, pp. 224-229). Various agonists or antagonists that can bind to RXRs are known (examples of the agonists include HX600 described in Japanese Patent Unexamined Publication (Kokai) No. 1′-59951 and the like, and examples of the antagonists include HX603 described in the same publication and the like). However, whether or not an organ can be formed from undifferentiated cells by using a ligand that binds to RXR has not yet been known so far.